African trypanosomiasis is lethal if not treated and its incidence is increasing. Existing therapies are antique by contemporary standards and no vaccine is available. The DNA topoisomerases are enzymes essential for nucleic acid biosynthesis and cell survival and are proven targets for clinically vulnerable anti- infective and antitumor drugs. Previous work from this lab has established that topoisomerase inhibitors cause dramatic lesions in nuclear and mitochondrial (kinetoplast) DNA which are directly proportional to cell killing. This is an ongoing project to examine the DNA topoisomerases as targets for antitrypanosomal drug development, and as essential enzymes in DNA metabolism and cell survival. The specific aims are two. First is to isolate and characterize native type I enzymes from trypanosomes, and to define the phenotypic consequence in trypanosomes of reducing or eliminating gene expression by knockout mutation or RNAi silencing. The second aim is to bring these basic molecular studies closer to the clinic by exploring the structure- activity relationship and selective toxicity of known topoisomerase IB poisons against trypanosomes in vitro, and by screening a defined chemical library to identify new topoisomerase poisons that are selectively toxic against trypanosomes. These studies take a multi-faceted, rational, and tangible approach to the development of much-needed new anti-trypanosomal chemotherapy.